Human serum albumin is a principal protein component present in plasma, it consists of a single chain polypeptide containing 585 amino acids and it has a molecular weight of about 66,000 Dalton (Da) (see, for instance, Non-Patent Document 1 given below). Principally, it has been known that human serum albumin plays a role to maintain the normal osmotic pressure of a blood and that it can also serve as a carrier for transporting a variety of substances appearing in the blood such as calcium ion, fatty acids, bilirubin, tryptophan and drugs or it can be linked with the foregoing substances to thus transport the same. Purified human serum albumin is used for the treatment of, for instance, hypoalbuminemia caused due to the loss of albumin through, for instance, surgical operations, hemorrhagic shock, burn or nephrotic syndrome.
Conventionally, human serum albumin has been prepared from the human plasma by the Cohn's low-temperature ethanol fractionation technique or by a method comprising the steps of preparing a human serum albumin fraction (the human serum albumin is fractionated in the fraction V) according to the same fractionation technique and then purifying the fraction while making use of a variety of purification methods. However, this method suffers from problems in that it is difficult to secure a sufficient quantity of a raw material therefor and that the resulting human serum albumin may be contaminated with pathogens and accordingly, there has been desired for the development of a technique for the preparation of the human serum albumin which is not derived from human plasma. As a method for solving such a problem, there have recently been developed techniques for using yeast fungal cells (see Non-Patent Document Nos. 2, 3 and 4 given below); Escherichia coli cells (see Non-Patent Document Nos. 5 and 6 given below); Bacillus subtilis (see Non-Patent Document 7 given below); or animal cells to produce human serum albumin.
In this regard, the resulting human serum albumin can in general be purified by any one of purification techniques currently used in protein chemistry such as the salting out, ultrafiltration, isoelectric precipitation, electrophoresis, ion exchange chromatography, gel filtration chromatography or affinity chromatography technique. In fact, the human serum albumin thus obtained contains a plurality of proteins such as biological tissues, cells and blood in an admixed condition and therefore, the human serum albumin is purified according to a complicated combination of the foregoing purification techniques. These methods have been applied to the method for the preparation of human serum albumin starting from a raw material produced according to the gene-recombination technique (see, for instance, Patent Document Nos. 1, 2 and 3 specified below).
It has been well-known that a human serum albumin is stable against heat-treatment in the presence of acetyl tryptophan and caprylic acid (see, for instance, Non-Patent Document 8 given below). Such heat-stability characteristics of the human serum albumin have been incorporated into the process for the preparation of the same for the purpose of deactivation of any protease present in the supernatant of a culture medium (see, for instance, Patent Document 4 specified below) and likewise used in a method for the sterilization of a final pharmaceutical preparation (see, for instance, Patent Document 5 specified below). The heat-treating method used in the production process can be considered to be useful in that it can process a large quantity of a human serum albumin-containing solution.
In most of cases, a large quantity of human serum albumin is administered to a patient in the aforementioned treatments and therefore, possible side effects of impurities present therein become an important problem as compared with a vaccine or other drugs administered in a small amount. For this reason, the human serum albumin prepared through a gene engineering technique should have a purity extremely higher than those required for a vaccine or conventional preparations containing human serum albumin originated from plasma. Moreover, it is necessary to establish a preparation method which permits the treatment of a large quantity of a raw material at low cost while taking into consideration stable supply of a human serum albumin to the market.                Patent Document 1: Japanese Patent No. 2,885,212;        Patent Document 2: JP-T-Hei 11-509525;        Patent Document 3: JP-A-Hei 6-100592;        Patent Document 4: JP-B-Hei 6-71434;        Patent Document 5: JP-A-Hei 7-126182        Non-Patent Document 1: Minghetti, P. P. et al., “Molecular Structure of the human albumin gene is revealed by nucleotide sequence within q11-22 of chromosome 4.”, J. Biol. Chem., 1986, 261:6747-6757;        
Non-Patent Document 2: Alan V. Quirk, Michael J. Geisow et al., “Production of Recombinant Human Serum Albumin from Saccharomyces cerevisiae”; Biotechnology and Applied Biochemistry, 1989, 11:273-287;                Non-Patent Document 3: Ken Okabayashi, et al., “Secretory Expression of the Human Serum Albumin Gene in the Yeast, Saccharomyces cerevisiae”; J. Biochem., 1991, 110:103-110;        Non-Patent Document 4: Richard G. Buckholz and Martin A. G. Gleeson “Yeast Systems for the Commercial Production of Heterologous Proteins”; Bio/Technology, 1991, 9:1067-1072;        Non-Patent Document 5: Lawn, R. M., “Construction of DNA sequences and their use for microbial production of proteins, in particular, human serum albumin”, “European Patent Appl.”, 1983, 73:646;        Non-Patent Document 6: Latta, L. et al., Synthesis and purification of mature human serum albumin from E. coli; Biotechnique, 1897, 5:1309-1314;        Non-Patent Document 7: Saunders, C. W. et al., Secretion of human serum albumin from Bacillus subtilis, J. Bacteriol., 1987, 169:2917-2925;        
Non-Patent Document 8: “Standard for Biological Preparations”, pp. 285-289, Published on October 10, in Showa 60 (1985), Incorporated Body: Association of Bacterial Preparations.